Studies were conducted to determine whether the production of various cytokines is associated with Mycoplasma pulmonis disease expression. Susceptible C3H/HeN and resistant C57BL/6N mice were inoculated intranasally with 107 CFU of virulent M. pulmonis UAB CT or avirulent M. pulmonis UAB T. Expression of genes for tumor necrosis factor alpha (TNF-α), interleukin 1α (IL-1α), IL-1β, 1L-6, and gamma interferon (IFN-γ) in whole lung tissue and TNF-α gene expression in bronchoalveolar lavage (BAL) cells was determined by reverse transcription-PCR using specific cytokine primers at various times postinoculation. In addition, concentrations of TNF-α, IL-1, IL-6, and IFN-γ were determined in BAL fluid and serum samples at various times postinoculation. Our results showed that there was a sequential appearance of cytokines in the lungs of infected mice: TNF-α, produced primarily by BAL cells, appeared first, followed by IL-1 and IL-6, which were followed by IFN-γ. Susceptible C3H/HeN mice had higher and more persistent concentrations of TNF-α and IL-6 in BAL fluid than did resistant C57BL/6N mice, indicating that TNF-α and possibly IL-6 are important factors in pathogenesis of acute M. pulmonis disease in mice. Serum concentrations of IL-6 were elevated in C3H/HeN mice, but not C57BL/6N mice, following infection with M. pulmonis, suggesting that IL-6 has both local and systemic effects in M. pulmonis disease.